1. Field of the Invention
The invention relates generally to mass spectrometry. The invention more specifically relates to a mass spectrometer having an ion source that permits high throughput sample analysis and a method for rapidly ionizing one or more samples in a mass spectrometer.
2. Background of the Invention
Mass spectrometers are instruments used to analyze ions with respect to their mass to charge ratio (m/z) to determine the chemical structures of molecules. In these instruments, molecules become positively or negatively charged in an ion source; the resulting ions are then transported to a mass analyzer, which measures their mass/charge (m/z) ratio. Mass analyzers come in a variety of types, including magnetic sector field (B), combined (double-focusing) electrostatic and magnetic field (EB), quadrupole (Q), ion cyclotron resonance (ICR), quadrupole ion trap (IT), and time-of-flight (TOF) mass analyzers.
The analysis of ions using a time-of-flight mass spectrometer (TOFMS) is, as the name suggests, based on the measurement of the flight times of ions as the ions travel through a field free region. Ions are typically extracted from an ion source in small packets and accelerated to a constant kinetic energy in a high voltage field. The velocities of the ions at constant kinetic energy varies according to the mass-to-charge ratio of the ions. Lighter ions will have greater velocities and will arrive at a detector earlier than high mass ions. Determining the time-of-flight of the ions through the drift chamber of the TOF mass analyzer to the ion detector permits the determination of the masses of different ions.
In a TOFMS instrument, single-charged molecular and fragment ions formed in the source are accelerated to a kinetic energy:
xe2x80x83eV=xc2xd mv2xe2x80x83xe2x80x83(eq. 1)
where e is the elemental charge, V is the potential across the source/accelerating region, m is the ion mass, and v is the ion velocity. These ions pass through a field-free drift region with velocities given by equation 1. The time (t) required for a particular ion to travel across the drift region is directly proportional to the square root of the mass/charge ratio:
t=L(m/2eV){circumflex over ( )}0.5xe2x80x83xe2x80x83(eq. 2)
where L is the length of the ion flight path. Conversely, the mass/charge ratios of ions can be determined from their flight times according to the equation:
m/e=at2+bxe2x80x83xe2x80x83(eq. 3)
where a and b are constants which can be determined experimentally from the flight times of two or more ions of known mass/charge ratios.
Since the earlier concept of time-of-flight instrument described by Stephens (W. E. Stephens, Bull. Am. Phys. Soc. 21, p22 (1946), a number of strategies have been employed for improving the performance of mass spectrometers. Significant improvement has been achieved, especially in mass resolving power. These strategies include the use of space and time-lag focusing (a method for spatial and energy focusing), as described by Wiley and McLaren (Wiley and McLaren, Rev. Sci. Instrum 26 (1955) 1150), the use of ion reflectron by Mamyrin et al. to compensate for energy spread of ions of equal mass-to-charge ratio (Mamyrin et al., Sov. Phys. JETP 37 (1973) 45), and the use of orthogonal ion extraction as described by Guilhaus et al. (U.S. Pat. No. 5,117,107) for increasing instrument duty cycle and reducing the influence of the initial energy spread of ions. A commercial instrument employing Wiley-McLaren technology was supplied by Bendix Corporation (Model NA-2) and later by CVC Products (Model CVC-2000).
A time-of-flight mass spectrometer can also be used as a basic platform for a tandem mass spectrometer. A tandem mass spectrometer is an instrument that combines two or more mass analyzers in a single instrument (MS/MS, MS/MS/MS, etc.) in combination with an ion-molecule collision cell. Tandem mass spectrometers have a particular advantage for structural analysis in that the first mass analyzer (MS1) can be used to measure and select a molecular ion from a mixture of molecules, while the second mass analyzer (MS2) can be used to analyze the fragment ions derived from the selected molecular ion. The fragment ions usually are produced in collision cell via collisional induced dissociation (CID).
The most remarkable advantage of time-of-flight mass spectrometer is theoretically unlimited mass range it can detect. This renders TOFMS instruments particularly powerful for biochemical applications. However, until the early 1970""s, most published TOFMS instruments employed electron impact ionization (EI). This ionization technique was limited only to volatile samples. The ionization of biological samples was made possible with secondary ion mass spectrometry (SIMS). SIMS was used to analyze peptides (Benninghoven et al., SIMS V, Springer Series in Chem. Phys. 44 (1986)). Other technologies useful in assessing biological samples include fast atom bombardment (FAB) (Chait et al., Int. J. Mass Spectrom. Ion. Phys. 40 (1981) 185),252Cf plasma desorption (McFarlane et al., Science 191 (1976) 920), laser-desorption ionization (LDI) (Hillenkamp et al., Appl. Phys. 8 (1975) 341), and electrospray ionization (ESI) (Fenn et al., Science 246 (1989) 64). The use of a reflectron in a laser microprobe instrument is described by Hillenkamp et al. (Appl. Phys. 8 (1975) 341). An instrument of similar design using LDI was produced by Leybold Hereaus as the LAMMA (LAser Microprobe Mass Analyzer). Cambridge Instruments produced a similar instrument called the Laser Ionization Mass Analyzer. Grotemeyer et al. (Org. Mass Spectrom. 22 (1987) 758) have used an instrument employing two lasers. The first laser is used to ablate solid samples, while the second laser forms ions by multiphoton ionization. A similar instrument has been manufactured commercially by Bruker.
An important category in LDI is so called matrix assisted laser desorption ionization (MALDI) technique described by Tanaka et al. (Rapid Commun. Mass Spectrom. 2 (1988) 151) and Karas et al. (Anal. Chem. 60 (1988) 2299). In MALDI, a sample (analyte) is mixed with an excess solution of matrix such as nicotinic acid and dispersed on an electrically conductive sample plate. The matrix absorbs the energy from a short laser pulse and produces a gas plasma, resulting in vaporization and ionization of the analyte. The combination of MALDI technique and TOFMS forms a powerful platform for analyzing biological samples such as DNAs, RNAs, peptides and proteins. Using MALDI-TOFMS biological samples of molecular weight range from several thousands to several hundred-thousand Dalton have been successfully ionized and analyzed.
In tandem instruments, fragmentation of the selected molecular ions to form fragment ions is induced in the region between the two mass analyzers. In one typical method of inducing fragmentation known as collision induced dissociation CID, the selected molecular ions are introduced into a collision chamber filled with an inert gas. The collisions of the ions and the inert gas to yield fragment ions may be carried out at high (5-10 keV) or low (10-100 eV) kinetic energies, or may involve specific chemical (ion-molecule) reactions. Other methods of inducing fragmentation include surface induced dissociation (colliding ions with surfaces to induce fragmentation), electron induced dissociation (using electron beams to induce fragmentation), or photodissociation (using laser radiation to induce fragmentation). The molecular ions may optimally dissociate at specific chemical bonds. The mass/charge ratios of the resulting fragment ions are used to elucidate the chemical structure of the molecule. It is possible to perform such an analysis using a variety of types of mass analyzers including TOF mass analyzers. The use of tandem mass spectrometers, such as a TOFMS-TOFMS combination or quadrupole-time-of-flight mass spectrometer (Q-TOF), when utilized with a collision cell has enabled the elucidation of the structure of large molecules including many biological compounds. Other LDI methods include preparing analytes on a modified silicon substrate (Wei et al., Nature 399 (1999) 243) or a thin film substrate (McComb et al., Rapid Commun. Mass Spectrom. 11 (1997) 1716).
Another strength of TOFMS instruments is the ability to determine the exact molecular weight of ions. Many of the commercial instruments combining ESI-TOFMS or MALDI-TOFMS are able to resolve the molecular weight to less than ten parts per million. Such mass determination accuracy is essential for peptide mapping and protein identification through database searching. In combination with laser ionization, LDI-TOFMS also achieved a high duty cycle of the detection. A large portion of ions produced with laser ionization can be ultimately detected since both of are ionization and extraction are inherently pulsed.
Moreover, TOF mass analyzers are very fast. Usually, the mass-to-charge ratio of a relatively large molecule can be determined in less than one millisecond. According to equation 2, a molecule of 100,000 Dalton can be recorded in 320 microseconds using a TOFMS with 2 meter drift path and 20 kV acceleration. Consequently, TOFMS is a primary choice for high-throughput mass analysis. In protein analysis, identification of large quantity of samples often is required on a day-to-day basis operation. In MALDI-TOFMS, efforts to increase throughput has been made by increasing the frequency of laser pulses (Loboda et al., Rapid Commun. Mass Spectrom. 14(2000) 1047).
FIG. 1 illustrates a laser desorption/ionization mass spectrometer as known in the art. In FIG. 1, a timing control circuit 100 activates a laser generator 102. A short laser pulse is focused onto a sample 104 carried on a sample plate 106 to desorb and ionize the analyte from the sample plate 106 surface. At the same time or after a short delay time (Colby et al., Rapid Commun. Mass Spectrom. 8 (1994) 865) a high voltage pulse, or extraction pulse, generated by an extraction pulse circuit 120 is applied to the sample plate 106 to generate a high electric field between sample plate 106 and electrode R1108, accelerating the ions via electrode R2110 towards a TOF mass analyzer 112. The ions travel through the TOF mass analyzer 112 and are recorded by an ion detector/preamplifier 114 and a data acquisition system 116. The spectral data obtained are then stored in a digital storage system 118 for future analysis. Neglecting the time needed for accelerating the ions from the ion extraction device (sample plate 106, electrode R1108, and electrode R2110) and delays in the electronic circuit, the analysis time, i.e., the flight time of ions, is given by Eq. 2, above. Generally, the flight time is less than 1 millisecond even for very large biomolecules.
A drawback of such a conventional MALDI-TOF instrument is that it is relatively xe2x80x9cslowxe2x80x9d in comparison with other ionization techniques. Most commercial laser generators deliver laser pulses between 1 to 100 Hz, typically 10 to 20 Hz. Normally, an accumulated ion signal of 100 laser shots is needed to obtain a spectrum with an adequate signal to noise ratio. Using a laser pulse of 10 Hz, this leads to an analysis time of 10 seconds for each sample, not including sample preparation and sample position. In comparison, TOFMS is capable of analyzing the ions in much higher speed. For a typical MALDI-TOF instrument using an acceleration voltage of 20 kV and a flight path of 2 meters, only approximately 320 xcexcs is needed for recording of a spectrum of mass up to 100,000 Daltons. Considering only the speed of TOFMS, the time required for accumulating 100 ion pulses is only 32 ms, which is more than 300 times faster than that can be delivered by the commercial laser generators.
It would be particularly useful to provide a mass spectrometer in which the mass analyzer is operated at or near its maximum rate of throughput. Thus, it would be advantageous to have an ion source capable of delivering ion packets to the mass analyzer more frequently.
Accordingly, a high-throughput laser desorption/ionization (LDI) mass spectrometer has been developed and is described herein. The mass spectrometer employs an ion source which comprises a plurality of lasers firing in tandem at one or more samples to increase the rate at which ion packets are generated by the ion source. The ion source is in operable association with a mass analyzer, preferably a time-of-flight mass analyzer, to provide the ion packets to the mass analyzer. The ion source may be used in other types of mass spectrometer instruments, e.g. Fourier transform ion cyclotron resonance (FT-ICR) instruments or quadropole-time-of-flight (QTOF) instruments. Timing circuitry associated with the ion source is used to control the firing of each laser and the application of an extraction pulse to deliver the ion packets to the mass analyzer. The generation of the extraction pulse is generally initiated simultaneously with the firing of the laser or within a short period of time thereafter. The samples are arranged on a sample plate which is preferably mounted on a moveable stage. Multiple lasers may be focused on a single sample, or each laser may be focused on a separate sample. When the sample(s) has been analyzed, the moveable stage may be advanced to bring the next samples online.
Also described is a method for performing high-throughput LDI mass spectrometry by using multiple lasers firing in tandem to generate ion packets in tandem and supplying the ion packets to a mass analyzer in a mass spectrometer. In the method, each firing of a laser results in an ion packet, which is analyzed in the mass analyzer prior to the firing of the next laser. When each laser has fired, the cycle starts over with the first laser.